History of Slavery in the USA
History of Slavery in the USA 129 137
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Journal of Biochemical and Biophysical Methods 16 1988 2740 27 Elsevier BBM 00653 In the absence of catalytic metals ascorbate does not autoxidize at pH 7 ascorbate as a test for catalytic metals Garry R Buettner GSF Research Center Institut fur Strahlenbiologle D8042 Neuherberg ERG Received 12 November 1987 Accepted 22 January 1988 Summary Trace amounts of adventitious transition metals in buffer solutions can serve as catalysts for many oxidative processes To fully understand what role these metals may play it is necessary that buffer solutions be catalytic metal free We demonstrate here that ascorbate can be used in a quick and easy test to determine if nearneutral buffer solutions are indeed catalytic metal free In buffers which have been rendered free of catalytic metals we have found that ascorbate is quite stable even at pH 7 The firstorder rate constant for the loss of ascorbate in an airsaturated catalytic metal free solution is less than 6gtlt107 s 1 at pH 70 This upper limit appears to be set by the inability to completely eliminate catalytic metal contamination of solutions and glassware We conclude that in the absence of catalytic metals ascorbate is stable at pH 7 Key words Ascorbate Iron Copper Autoxidation Introduction Trace amounts of adventitious transition metals which are naturally present in buffer solutions have been recognized as presenting many problems for researchers 1 17 These trace levels of transition metals are able to catalyze reactions such as ascorbate oxidation 1416 and the metalcatalyzed HaberWeiss reaction 11 Thus for experiments in which low concentrations of catalytic metals are an important determinant of the result these metals must be sequestered in an inactive form or removed from the reaction mixture While ascorbate readily autoxidizes Correspondence address GR Buettner NIEHS POB 12233 RTP NC 27709 USA Abbreviations Abs dAbsorbancedr AHZ ascorbic acid pK1 42 pK2 116 21 AH ascorbate monoanion AZ39 ascorbate dianion AT ascorbate radical mAU milliabsorbance units 1 cm path 0165 022X880350 1988 Elsevier Science Publishers BV Biomedical Division 28 catalytic transition metals such as copper or iron are required for this oxidation to proceed with an appreciable rate at acid or neutral pH 11618 20 Removal of these metals from buffer salt solutions significantly slows this process 16 We demonstrate that in the absence of catalytic metals ascorbate does not autoxidize at pH 7 Thus ascorbate can be used as a test for the presence of adventitious catalytic metals in buffer salt solutions We present here a quick and easy method by which nearneutral buffer salt solutions can be checked for their catalytic metal activity In addition we offer a reinterpretation of some of the ascorbic acid literature in which contaminating catalytic metals were present Materials and Methods Ascorbic acid lot analysis indicates 0000270 Fe sodium monohydrogen phos phate lot analysis indicates 0000370 Fe sodium dihydrogen phosphate lot analy sis indicates 0001 Fe and Tris trishydroxymethylaminomethane were from JT Baker Iminodiacetic acid TrisHCl and conalbumin Type IV were from Sigma Chelex 100 200400 mesh sodium form was from BioRad Laboratories Water used in solution preparations was purified with a Milli Q water system resistivity 18 M52 cm All reagents were used as received except where otherwise noted Absorbance measurements were done with a PerkinElmer Lambda 5 UV VIS spectrophotometer Each ascorbate sample in a standard 1 cm quartz cuvette was continuously monitored at 265 nm For the standard test ascorbic acid is prepared as a 0100 M stock solution 10 ml using high purity water such as is produced by the MilliQ water system This solution is colorless having a pH of z 2 It is stored in a volumetric flask with a tightfitting plastic stopper thus oxygen is kept from the solution during longterm storage As the solubility of oxygen in airsaturated water is 2025 mM the solution will become anaerobic with loss of lt 1 of the original ascorbate We have found that the solution can be kept for several weeks without significant loss of ascorbate due to the low pH and lack of oxygen The appearance of a yellow color is an indication of ascorbate deterioration We avoid the use of sodium ascorbate as it invariably contains a significant quantity of oxidation products as evidenced by the yellow color of the solution In a typical test z 35 pl of the ascorbate stock is added to 3 ml of the buffer salt solution to be tested This results in an initial ascorbate absorbance of 1520 at 265 nm The molar extinction coefficient of ascorbate used here is 14500 Mquot1 cm 1 at 265 nm However values ranging from 7500 to 20 400 M 1 cm 391 have been reported 21 The ascorbate absorbance at 265 nm is followed for 15730 min When possible buffer solutions are prepared by weighing out the appropriate amounts of buffer salts for a 10 M stock solution that will produce the desired pH upon dilution to the working concentration This avoids the use of acids or bases for pH adjustment which may be a source of additional adventitious catalytic metals 29 Chelex 100 treatment for the removal of contaminating metals can be accom plished using the batch method the dialysis method or the traditional column method In the batch method the resin is washed with portions of the buffer solution to be treated then rinsed with high purity water The washed resin is then added to the solution using 510 ml of resin per liter of solution and stirred overnight shorter times can be used if more resin is used After any necessary pH adjustments and additional stirring the resin is usually filtered from the buffer However it may be left in the solution and allowed to settle to the bottom before withdrawing portions for use being sure to check for suspended resin In the dialysis method 7 g of washed Chelex 100 resin is suspended in z 9 ml of the buffer The end of a disposable 5 ml plastic pipette tip is enlarged and used to transfer the Chelex 100 suspension to a suitable length of boiled ViskingR or similar dialysis tubing The cleaned sack is placed in 250 500 ml of buffer and stirred overnight The dialysis sack may be left in the solution to help overcome problems with catalytic metal enrichment from the glassware and atmosphere The dialysis method provides a simple way to easily remove the Chelex 100 from the solution For experiments in which the catalytic activity of lt 1 uM CuII was examined stock solutions of 0060 mM pH 15 and 060 mM pH 15 CuCl2 were prepared The order of reagent addition to the cuvette containing the chelexed buffer solution was CuII stock followed by shaking then enough ascorbate to produce a solution with an absorbance of 18 i 01 at 265 nm 1 cm corresponding to a concentration of 125 i 7 11M The loss of ascorbate absorbance at 265 nm was followed for z 5 min As the loss was nearly linear in this time the slope was determined for the time of approximately 1 4 min after the introduction of the ascorbic acid For the iron experiments a stock solution of 060 mM FeCl3 was prepared in dilute HCl pH 20 Standardization of the iron concentration was accomplished using EHPG ethylenediamine NN39bis22hydroxyphenyl acetic acid 2223 Experiments were performed in the same manner as with the copper except the loss of ascorbate was followed for 15 min Adsorption of catalytic metals on the cuvette surfaces proved to be a significant limiting factor for achieving a near 0 loss pH 70 phosphate of ascorbate in the typical 15min test period This problem was especially frustrating after experiments in which micromolar FeIII or CuII was added to the reaction mixture We found the most effective way to reduce this effect was simply to fill the cuvettes with dilute HCl 002 M and let them stand overnight After running only a few samples in an acidsoaked cuvette significant loss of ascorbate greater than 1 in chelexed 50 mM phosphate pH 70 was often observed even if the cuvette was copiously washed with dilute acid and purified water Dialysis against conalbumin was done as outlined by Gutteridge 17 05 g conalburnin and 40 mg NaHCO3 were dissolved in 10 ml of the buffer and transferred to the boiled ViskingR dialysis tubing After thorough washing to remove any protein that might have been spilled on the outside surface the sack was placed in 300 ml of buffer and stirred overnight z 4 C 30 Results Ascorbate test As seen in Table l ascorbate autoxidation provides a simple test to determine if buffer solutions are catalytic metal free The per cent ascorbate lost ranged from 5 11 in untreated phosphate buffer to 06 or less after Chelex 100 treatment The changes observed in Tris or Krebs Ringer phosphate buffer were less dramatic but at pH 74 ascorbate is a good indicator for catalytic metal content provided clean cuvettes are used However at pH 90 or above where A2 will be a significant species ascorbate is of little value as an indicator TABLE 1 ASCORBATE LOSS IN BUFFER SOLUTIONS Solution Treatment amp additions 9 Ascorbate lost in 15 min quot 50 mM acetate buffer pH 50 50 mM acetate buffer pH 50 50 mM phosphate C pH 60 50 mM phosphate pH 60 50 mM phosphate pH 70 50 mM phosphate pH 70 50 mM phosphate pH 70 50 mM phosphate pH 70 50 mM phosphate pH 70 50 mM phosphate pH 70 50 mM phosphate pH 70 50 mM phosphate pH 70 50 mM phosphate pH 70 50 mM phosphate pH 78 50 mM phosphate pH 78 Krebs Ringer phosphate pH 74 b KrebsRinger phosphate pH 74 50 mM Tris pH 74 50 mM Tris pH 74 50 mM Tris pH 90 50 mM Tris pH 90 none Chelex 100 none Chelex 100 none none 50 11M EDTA Chelex 100 Chelex 100 dialysisconalbumin dialysisconalbumin 50 11M EDTA dialysisChelex 100 Chelex 100 1 11M Cull Chelexl 11M CuII 50 11M EDTA none Chelex 100 none Chelex 100 none Chelex 100 none Chelex 100 11 01 b 01 009 48 08 03 02 107 d 27 11 04 05 005 lt 005 c 004 11 06 04 004 05 05 27 25 f 04 005 84 25 06 02 11 01 05 01 18 02 05 03 36 05 31 07 a b r a n overnight m w The results quoted are the median of at least four trials Errors quoted are standard deviations Phosphate implies a NaH2P04Na2HPO4 buffer prepared as in Materials and Methods This may appear to be at odds with the results reported in 16 but different sources and grades of phosphate were employed Results obtained from the very first test using cuvettes that had been soaked in HCI 002 M This is the loss in 5 min rather than 15 min of the standard test Cafree KrebsRinger solution contained 120 mM NaCl 5 mM KCl 13 mM MgCl2 and 16 mM sodium phosphate buffer pH 74 31 Iron and copper Iron and copper are common trace level contaminants in reagents From the lot analyses provided by Baker Chemical the FeIII concentration in the 50 mM pH 70 phosphate buffer is estimated to be 07 MM In Fig 1 we see that the rate of ascorbate loss is directly proportional to the iron concentration EDTA increased the catalytic activity of FeIII as is well known 1118720 However CuII is a much more effective catalyst than FeIII or FeIIIEDTA In comparing Fig 1 and 2 we see that CuII is z 80 times more effective than FeIII and z 20 times more effective than FeIII EDTA 50 mM phosphate buffer pH 70 But in contrast to FeIII the addition of EDTA to the copper ascorbate system stops the oxidation of ascorbate See Table 1 The inclusion of EDTA in the untreated phosphate buffer 50 mM pH 70 ascorbate system slowed the oxidation in the standard test from 107 loss Abs 128 mAUmin to 11 Abs 13 mAUmin From this observation we conclude that the majority of the catalytic activity observed is not due to iron Using the FeIIIEDTA data presented in Fig 1 the 11 loss of ascorbate indicates an FeIII concentration of z 03 pM which is in remarkable agreement with that estimated from the Baker analyses If we subtract the catalytic activity due to FeIII in the untreated buffer from the total activity Abs 128 03 125 mAUmin we have the catalytic activity Fe III Ascorbate 25gt FeIIIEDTA 15 12 E 3 lt2 E m 2 Penn 5 v 0 1 1 1 1 1 O 20 40 60 Fem pM Fig l FeIII and FeIIIEDTA catalyzed oxidation of ascorbate 125 i 7 uM in Chelex lOOtreated 50 mM phosphate buffer pH 70 O FeIII A FeIII EDTA The ordinate represents the rate of loss in the 265 nm absorption of ascorbate in units of milliabsorbance unitsmin 1 cm path Cu IDAscorbate 100 O O thsmAU min391 20 o l 05 Cu 11 LJM Fig 2 CuII catalyzed oxidation of ascorbate 125i7 uM in Chelex IOU treated 50 mM phosphate buffer pH 70 The ordinate represents the rate of loss in the 265 nm absorption of ascorbate in units of milliabsorbance unitsmin 1 cm path The addition of 50 11M EDTA to a 1 pM Cull solution reduced the rate of ascorbate loss from 96 to 04 mAUmin quot 1 10 due to copper Then from the data of Fig 3 we can estimate that the concentration of copper in the untreated phosphate buffer is z 013 uM Unfortunately the Baker lot analyses did not include copper to allow a comparison After Chelex 100 treatment of the pH 70 phosphate buffer the loss of ascorbate in the standard test is reduced to 04 Abs 05 mAUmin However in trials in which the standard test was performed as the first sample in a cuvette which had been soaked overnight in dilute HCl this value was 01 Abs 012 mAUmin or less the median value being 005 From the values for the rate of ascorbate loss presented in Fig l and 2 we can set an upper limit of 01 uM using 01 loss for the concentration of Felll in Chelex lOOtreated 50 mM phosphate buffer pH 70 For copper this limit is on the order of 0001 uM These concentrations must be considered as estimates as we are at the limit of detection by this technique We are near or in the noise level of the experiment due to additional phantom sources of catalysis such as Fe or Cu in the ascorbate metals on the glassware and pipette surfaces dust in the air and the like Dialysis of the 50 mM pH 70 phosphate buffer against conalbumin or Chelex 100 reduced the catalytic metal content of the buffer as demonstrated by the standard ascorbate test see Table 1 However residual catalytic activity was observed in the buffer dialyzed against conalbumin The addition of 50 MM EDTA 33 to this buffer reduced this 11 loss to 04 Thus iron appears to be removed from the solution but some residual copper remains 10 15 nM Dialysis against Chelex 100 reduced both the copper and iron catalytic activity yielding the same results in the standard test as the Chelex 100 batch method Kinetics The oxidation of ascorbate excess oxygen has been found to be firstorder with respect to the concentration of ascorbate anion in the pH range 26 19 At pH 70 the dominant species for ascorbate will be AH 999 with low concentrations of AH2 01 and Az 0005 Under these conditions the pseudofirstorder rate equation can be written as dTAdtk2AH where TA is the total concentration of all ascorbate species In the experiments with acidsoaked cuvettes and chelexed pH 70 phosphate buffer the lowest consistent loss median of eight samples of ascorbate in the standard 15 min test was 005 This yields an upper limit for k2 of 6 X 10397 s For the iron and copper catalyzed oxidation of ascorbate excess oxygen the rate of loss of ascorbate in pH 70 phosphate buffer may be written as dTAdt ccatAH CuII or FeIII From the data of Figs 1 and 2 kcat for FeIII 10 kcal for FeIII EDTA 42 and kcal for CuII 880 M 1 s 1 20 C It should be kept in mind that ferric and cupric ions will undergo hydrolysis at pH 7 Thus these rate constants do not represent rate constants for FeIIIaq or CuIIaq but rather are rate constants for the analytical concentration of FeIII and CuII without regard to the concentra tion of the various hydrolysis species present Discussion Tables 2 and 3 are a compilation of the levels of contaminating iron and copper found in commonly used reagents Unless care is taken to reduce these naturally occurring concentrations or the metals are complexed in a catalytically inactive form these catalytic metals can present serious problems in the interpretation of experimental results 1 17 These problems can in principle be overcome by use of ionexChange resins such as Chelex 100 or by dialysis such as used by McDermott et a1 4 and modified by Gutteridge 17 However each technique has its problems Chelex 100 The use of Chelex 100 to demetal acids or bases will result in an increase in the catalytic metal concentration 13 see Table 2 This is to be expected as strong acid 34 TABLE 2 TRACE IRON CONCENTRATIONS 1N SOLUTIONS Reagent Treatment FettM Ref 200 mM acetate pH 55amp65 none 101 278 4 200 mM acetate pH S5amp65 ChelexlOO 0014 h 4 200 mM acetateceruloplasmin pH 65 helexlOO 04 quot 4 200 mM acetateceruloplasmin pH 65 ChelexlOO 00070021 4 amp dialysis against transferrin 50 mM phosphate pH 70 none 07 Here 1 50 mM phosphate pH 70 none 03 Here C 50 mM phosphate pH 70 ChelexIOO lt 01 Here 100 mM phosphate pH 74 none 1 t 150 mM phosphate pH 74 none 22 7 150 mM phosphate pH 74 ChelexIOO 0055 7 20 mM phosphate pH 74 none 02 14 675 mM phosphate pH 7440 mM KC1 none 97 l94 12 100 mM Tris pH 74 none 17 17 100 mM Tris pH 74 dialysis 0 17 against conalbumin 100 mM KC1 none 25 13 500 mM sodium formate none 93 12 500 mM urea none 2 12 500 mM thiourea none 31 12 100 mM ascorbic acid none 22 13 20 mM ascorbic acid none 43 12 30 H202 none 18 91 13 10 mM EDTA none 07 13 10 mm EDTA ChelexlOO gt 75 13 50 mM EDTA none 7189 12 10 mM DETAPAC 5 none 18 13 10 mM DETAPAC Chelex lOO 144 247 13 4 NaOH none 73 13 4 NaOH Chelex100 193 13 5 HCl none 36 13 5 HCl ChelexlOO 114 13 05 mM xanthine none 15 13 500 mM mannitol none 0 l 12 400 mM sucrose none 1 2 2 21 Uml xanthine oxidase none 125 h 13 85 mgml hyaluronic acid none 103445 12 Water doubledistilled 0 39 12 Water ChelexIOO 0 r 12 a Most of the values summarized here fall within the range expected when making a solution from reagent grade stock See for example the analyses provided by suppliers such as IT Baker Fluka AG or Merck We have made this estimate from the data presented in 4 assuming an initial iron concentration of 20 MM 5 Fe was used for the determination Its concentration decreased to 0075 of the concentration before ChelexIOO treatment We have made this estimate from the data presented in 4 assuming an initial iron concentration of 20 pM nge was used for the determination Its concentration decreased to 2 of the concentration before ChelexlOO treatment er a TABLE 2 continued Calculated using the lot analysis supplied by Baker assuming no contribution from the water 5 Determined from the FeIII EDTA data of Fig 1 f To be interpreted as below the limit of detection which was not given A statistical analysis of the limited data presented in 30 suggests that 05 075 11M Fe is the approximate lower limit of detection by the bleomycin assay DETAPAC DTPA or diethylenetiiaminepentaacetic acid This is a shockingly large value however it must be kept in mind that 21 unitsml is on the order of 1000 times more than would be used in a typical experiment in which xanthine oxidase is used as a means to generate superoxide In addition xanthine oxidase contains four irons as FezSZ groups as part of its catalytic site This iron rather than adventitious iron could easily account for the bulk of the iron observed in this atomic absorption analysis The authors gave no indication as to whether any correction was made for this intrinsic iron a rrn TABLE 3 TRACE COPPER CONCENTRATIONS IN SOLUTIONS Reagent Treatment Cu MM 3 Ref 1 M KNO3 pH 60 none 0035 10 1 M KNO3 pH 60 Chelex 100 ND b 10 1 M KN03 pH 60 APDTC C ND 10 1 M NaCl pH 60 none 005 10 1 M NaCl pH 60 Chelex 100 ND 10 1 M NaCl pH 60 APDTC ND 10 1 M KHzPOA pH 60 none 03 10 1 M KHZPO4 pH 60 Chelex 100 004 10 1 M KH2P04 pH 60 APDTC lt 003 10 50 mM phosphate buffer pH 70 none 013 Here d 50 mM phosphate buffer pH 70 Chelex 100 239 0001 Here d 400 mM sucrose none 008 03 2 100 mM acetate pH 54 or 60 Chelex 100 lt 016 3 50 mM phosphate pH 60 or 74 Chelex 100 lt 016 3 1 M glycine buffer pH 89 Chelex 100 ND 9 a Most of the values summarized here fall within the range expected when making a solution from reagent grade stock See for example the analyses provided by suppliers such as IT Baker Fluka AG or Merck ND not detected using atomic absorption APDTC complexing with ammonium pyrrolidinedithiocarbamate followed by extraction with chloro form The data of Figs 1 and 2 were used to make this estimate c and base are used to regenerate Chelex 100 In addition strong complexing agents such as EDTA are also able to extract metals from Chelex 100 resin thus it is futile to attempt to demetal such reagents with Chelex 100 13 It follows logically that chelating agents should not be present in buffer solutions when they are to be demetalled with Chelex 100 See Bio Rad Laboratories product information bulletin 2020 36 Chelex 100 provides a means to demetal simple salt and buffer solutions However attempts to remove adventitious metals from protein solutions can present problems For example Binder et al 5 found that Chelex 100 formed a complex with coppercollagen The collagen could only be eluted from the Chelex 100 column by acidifying the resin In addition Buettner 24 observed that Chelex 100 formed a complex with cytochrome c at neutral pH removing it from solution Thus proteinmetalChelex 100 complex formation must be considered when at tempts are made to remove metals from protein solutions Biological effects are also possible when using Chelex IOUtreated solutions Rayment and Andrew 10 observed retardation of plant growth when Chelex IOUtreated solutions were used in their studies They attributed this to free iminodiacetic acid which can form when Chelex 100 stands for some time Removal of free iminodiacetic acid from the resin prior to its use eliminated this effect Dialysis Dialysis is an effective method of removing metals from buffer and protein solutions Transferrin or conalbumin will be effective in removing iron but some copper may remain and it is possible that protein contaminants may be introduced into the solution Dialysis against Chelex 100 will remove both copper and iron providing a means of circumventing the possible introduction of protein contami nants as well as avoiding formation of proteinmetalChelex 100 complexes In our experiments Chelex 100 was placed in the dialysis sack to demetal the buffer However to de metal a protein solution it may be more appropriate to place the protein in the dialysis sack and dialyze it against a suspension of Chelex 100 This method was used effectively by Harris et al 8 in their hyaluronic acid depolymeri zation studies Glassware Buffer solutions can also undergo catalytic metal enrichment during storage 812 personal observations and frustrations We have found as have Harris et al 8 that keeping Chelex 100 in contact with buffer solutions during storage overcomes this problem However the ascorbate test provides a quick and easy method to monitor for this potential problem Limits of the ascorbate test The ascorbate test for catalytic metals is most effective in the nearneutral or slightly acid pH range where AH is the dominant species At higher pH values gt 8 AZ becomes a significant species Because it rapidly autoxidizes the sensitivity of the test is lost Likewise at low pH values AH2 will dominate and it autoxidizes too slowly 20 to provide a sensitive test In addition high concentra tions of CI in the media will also reduce sensitivity because high chloride concentrations inhibit low concentrations can accelerate the coppercatalyzed oxidation of ascorbate 2526 Large variations in the loss of ascorbate in untreated buffers should be anti cipated depending on the source and grade of the chemicals and history of the 37 glassware Compare for example results reported in 163335 and here Thus removal of these catalytic metals is often essential for a comparison of experimental results from different laboratories or even within the same laboratory Reinterpretation of ascorbic acid literature The spontaneous oxidation of ascorbate anion at pH 70 ie in the absence of catalytic metals is quite slow We have set an upper limit of 6 X 10 7 s This limit may be the result of catalytic metals which were not removed from the solution by Chelex 100 treatment as well as possible catalytic metals on the cuvette surfaces Khan and Martell 19 have reported a value of 587 X 10 4 s 1 for the spontaneous oxidation of AH in 01 M KNO3 As no efforts were made to remove con taminating catalytic metals there was undoubtedly substantial CuII and FeIII present see Tables 2 and 3 Thus we believe this rate constant is grossly overestimated Using data from other studies the observed firstorder rate constant for the spontaneous oxidation of AH was found to be 2 X 10 6571 chelexed pH 70 phosphate buffer oxygensaturated 31 which is comparable to the value reported here In a study of copper and ascorbate oxidation in aqueous humor Fong et a1 29 have demonstrated that aqueous humor can accelerate or inhibit coppercatalyzed oxidation of ascorbate Those aqueous humor samples with high copper content 25 uM contained little or no ascorbate lt 01 mg while those with low copper 05 uM contained substantial ascorbate 22 30 mg However in the light of the data presented here it is clear they had substantial adventitious copper present in their buffer z 01 MM In most experiments they added 04 MM copper to the incubations Thus the actual copper concentration is 05 MM Although the overall conclusions are not changed by the high backround level of copper the exact interpretation of the data is altered and is consistent with z 01 MM adventitious copper Mishra and Kovachich have examined the inhibition of ascorbate oxidation by extracts from mammalian nervous tissue and serum 32 34 plant tissues 35 and microorganisms 36 Their assay medium was typically KrebsRinger phosphate buffer pH 74 Thus the autoxidation of ascorbate they observed is due to adventitious catalytic metals which are an uncertain element in their control experiments Without additional control experiments using demetalled medium the mechanism for the inhibition of ascorbate oxidation by their extracts cannot be deduced Another possible problem which can result from adventitious metals in buffer solutions is synergistic catalytic activity For example Matsumura and Pigman 37 found that copper and iron acted synergistically in the metalcatalyzed depolymeri zation of hyaluronic acid by ascorbate In a somewhat similar case Yamazaki 38 found that A7 generated by ascorbate oxidase will reduce cytochrome c However Weis 39 and references therein was unable to reproduce this result unless CuII was present He suggested that contaminating copper was present in the ascorbate oxidase used by Yamazaki Thus adventitious metals can alter reaction endpoints consequently altering conclusions 38 Superoxide and metal catalysis The generation of the very reactive hydroxyl radical in superoxidegenerating systems was first proposed to result from the reaction of O with HlO2 40 However it was soon recognized that iron played a role in this reaction 4142 Only trace amounts of iron the levels present in typical buffer solutions were required to alter the results observed in superoxidegenerating systems 11 Thus just as in ascorbate oxidation adventitious metals can alter the results observed in superoxidegenerating systems and must be removed to insure that baseline or control experiments are reliable standards for interpretation of experimental results Simplified description of the method and its applications An easy and rapid method using ascorbic acid is presented to determine if simple nearneutral buffer salt solutions are free of catalytic metals In the standard test approximately 35 ul of 0100 M ascorbic solution are added to 3 m1 of the buffer solution being tested The loss of ascorbate is monitored for 15 min using its 265 nm absorbance A loss of more than 05 of the ascorbate in this time indicates significant metal contamination The advantages of this method are 1 very low cost 2 short time of determination 3 it requires no specialized instrumentation and 4 it provides an easy and repeatable method to determine and report experimental conditions References 1 Barron ESG DeMeio RH and Klemperer F 1936 Studies on biological oxidations V Copper and hemochromogens as catalysts for the oxidation of ascorbic acid The mechanism of the oxidation J Biol Chem 112 625 640 Noguchi Y and Johnson MJ 1961 Citric acid fermentation of sugars purified with chelating resin J Bacteriol 82 5387541 Morel AG Aisen P and Scheinberg LH 1962 Is ceruloplasmin an ascorbate acid oxidase J Biol Chem 237 3455 3457 McDermott JA Huber CT Osaki S and Frieden E 1968 Role of iron in the oxidase activity of ceruloplasmin Biochim Biophys Acta 151 5417557 Binder G Altman KI and Forbes WF 1970 Complex formation of collagen with copper and its application to the quantitative determination of collagen Anal Biochem 37 129437 Kessler W 1987 Untersuchungen zu Aminosaure und Proteinoxidationen in EisenAscorbat und EisenAscorbatGSHSystemen hinsichtlich der Entstehung von Kohlenwasserstoffen sowie En zyminaktivierungen PhD Dissertation University of Tiibingen p 13 Poyer IL and McCay RB 1971 Reduced triphosphopyridine nucleotide oxidase catalyzed alter ations of membrane phospholipids J Biol Chem 246 263a269 Harris MJ Herp A and Pigman W 1972 Metal catalysis in the depolymerization of hyaluronic acid by autoxidants J Am Chem Soc 94 75707572 Jacobsen GB and Rodwell VW 1972 A Bacillus ribonucleic acid phosphodiesterase with associated 5 nucleotidase activity J Biol Chem 247 58115817 Rayment GE and Andrew CS 1972 Biological and chemical comparisons of methods for removing copper from macronutrient solutions used for plant growth investigations Plant Soil 36 547 559 Buettner GR Oberley LW and Leuthauser SWHC 1978 The effect of iron on the distribution of superoxide and hydroxyl radicals as seen by spin trapping and on the Superoxide dismutase assay Photochem Photobiol 28 693 695 N La A kit 0quot l 00 D p A O i i b 4 A p I LII O n l a 30 0 Lo 1 39 Wong SF Halliwell B Richmond R and Skowroneck WR 1981 The role of superoxide and hydroxyl radicals in the degradation of hyaluronic acid induced by metal ions and ascorbic acid J Inorg Biochem 14 127 134 Borg DC and Schaich KM 1984 Cytotoxicin from coupled redox cycling of autoxidizing xenobiotics and metals lsr J Chem 24 38 53 Sutton HC and Winterbourn CC 1984 Chelated ironcatalyzed OH formation from paraquat radicals and H202 Mechanism of formate oxidation Arch Biochem Biophys 235 106 115 Chiou SH Chang WC Jou YS Chung HM M and Lo TB 1985 Specific cleavages of DNA by ascorbate in the presence of copper ion or copper chelates J Biochem 98 1723 1726 Buettner GR 1986 Ascorbate autoxidation in the presence of iron and copper chelates Free Rad Res Commun 1 349353 Gutteridge JMC 1987 A method for removal of trace iron contamination from biological buffers FEBS Lett 214 362364 Grinstead RR 1960 The oxidation of ascorbic acid by hydrogen peroxide Catalysis by ethylen ediaminetetraaceto ironIII J Am Chem Soc 82 3464 3476 Khan MMT and Martell AE 1967 Metal ion and metal chelate catalyzed oxidation of ascorbic acid by molecular oxygen 1 Cupric and ferric ion catalyzed oxidation JAm Chem Soc 89 41764I85 Martel AE 1982 Chelates of ascorbic acid formation and catalytic properties In PA Seib and BM Tolbert Eds Ascorbic Acid Chemistry Metabolism and Uses American Chemical Society Washington DC pp 124 178 Lewin S 1976 Vitamin C Its Molecular Biology and Medical Potential Academic Press London Underwood AL 1958 Spectroscopic determination of iron with ethylenediaminediohydroxy phenyl acetic acid Anal Chem 30 33 47 Buettner GR 1987 The reaction of superoxide formate radical and hydrated electron with transferrin and its model compound FeIIIethylenediamine NN bis22hydroxyphenyl acetic acid as studied by pulse radiolysis J Biol Chem 262 11995 11998 Buettner GR Saran M and Bors W 1987 The kinetics of the reaction of ferritin with superoxide Free Rad Res Commun 2 369372 Mapson LW 1945 Influence of halides on the oxidation of ascorbic acid 2 Action of Cl on the cupriccuprous system Biochem J 39 228 236 Scaife JF 1959 The catalysis of ascorbic acid oxidation by copper and its complexes with amino acids peptides and proteins Can J Biochem Physiol 37 1049 1067 Weissberger A and LuValle JE 1944 Oxidation processes XVII The autoxidation of ascorbic acid in the presence of copper J Am Chem Soc 66 700 705 Weissberger A LuValle JE and Thomas DS Jr 1943 Oxidation processes XVI The autoxida tion of ascorbic acid J Am Chem Soc 65 1934 1939 Fong D Etzel K Lee PF Lin TYM and Lam KW 1987 Factors affecting ascorbate oxidation in aqueous humor Curr Eye Res 6 357 361 Gutteridge JMC Rowley DA and Halliwell B 1981 Superoxide dependent formation of hydroxyl radicals in the presence of iron salts Detection of free iron in biological systems by using bleomycindependent degradation of DNA Biochem J 199 263 265 Scarpa M Stevanato R Vigino P and Rigo A 1983 Superoxide ion as active intermediate in the autoxidation of ascorbate by molecular oxygen Effect of superoxide dismutase J Biol Chem 258 6695 6697 Kovachich GB and Mishra OF 1982 Inhibition of ascorbate autoxidation by a rat brain cortical factor Neurosci Lett 34 83 87 Misra OR and Kovachich GB 1983 Inhibition of ascorbate autoxidation by the dialyzed soluble fraction of mammalian nervous tissues Neurosci Lett 43 103 108 Kovachich GB and Mishra OF 1984 Stabilization of ascorbic acid and norepinephrine in vitro by the subcellular fractions of rat cerebral cortex Neurosci Lett 52 153 158 Mishra OR and Kovachich GB 1984 Inhibition of ascorbate autoxidation by a dialyzed heatdenatured extract of plant tissues Life Sci 34 2207 2212 Mishra OR and Kovachich GB 1984 Inhibition of the autoxidation of ascorbate and 40 norepinephrine by extracts of Clostridium butyricum Megaxphaera elsdenii and Escherichia 601139 Life Sci 35 849 854 Matsumura G and Pigman W l965 Catalytic role of iron ions in the depolymerization of hyaluronic acid by ascorbic acid Arch Biochem Biophys 110 526 533 Yamazaki I 1962 The reduction of cytochrome c by enzyme generated ascorbate free radical J Biol Chem 237 224 229 Weis W 1975 Ascorbic acid and electron transport Ann NY Acad Sci 258 l90 200 Beauchamp C and Fridovich I 1970 A mechanism for the production of ethylene from methional The generation of the hydroxyl radical by xanthine oxidase J Biol Chem 245 464141646 Fong KL McCay PB Poyer JL Keele BB and Misra HP 1973 Evidence that peroxidation of lysosomal membranes is initiated by hydroxyl free radicals produced during flavin enzyme activity J Biol Chem 248 77927797 Fong KL McCay PB and Poyer IL 1976 Evidence for superoxidedependent reduction of FeIII and its role in enzymegenerated hydroxyl radical formation ChemBiol Interact 15 77789
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